Preparation of cells
Using the Fresenius Kabi system, PBMCs were collected from each patient under electrocardiographic monitoring. The cells were cultured overnight, then adherent cells (including monocytes) and suspension cells (including lymphocytes) were collected separately.
Preparation of the DC vaccine
Isolated PBMCs were resuspended in a serum-free culture medium. The PBMCs were then seeded into a 175-cm3 flask to a concentration of 2-4×106/ml, divided among three bottles and then placed in a 37 ̊C, 5% CO2 incubator. After 2 hours of incubation, the 175-cm3 flasks were gently agitated; the suspension cells were discarded (or collected and used for culturing CIK cells), then washed 1-2 times with serum-free medium and, following addition of the DC medium, placed again in the 37 ̊C, 5% CO2 incubator. Autologous serum, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4 and tumor necrosis factors (all from PeproTech EC, Ltd., London, UK) were added on the 3rd day. The tumor antigen was added on the 5th day and the ripening factor was added following addition of the tumor antigen. DC cells were collected between the 7th and 8th days and then washed three times with saline.
Preparation of CIK cells
The culture bottles were pre-coated with a 5 μg/ml final concentration of CIK reagents before preparing the CIK cells, then placed in a 4 ̊C environment overnight and washed with phosphate- buffered saline 2-3 times on day 0. The CIK cells were resuspended in the initial CIK cell medium at a concentration of 2-3×106/ml and spread into a culture bag according to the number of cells. Autologous plasma was then added and cultured in a 37 ̊C, 5% CO2 incubator. IL-2, interferon 2γ and CD3 monoclonal antibody (100 μg/ul) were added using 5-ml disposable syringes with needles.
The DC vaccine and CIK cell therapy schedule was as follows: The day on which peripheral blood mononuclear cells (PBMCs) were collected was defined as day 0. On day 8, 1×107 DCs were infused intravenously once/week for 6 weeks, and on day 11, 1×109 CIK cells were infused intravenously once/day for 4 days.
DTH, QOL and adverse effects
A DTH skin test used to assess the immune response to cell therapy for all patients and the results were recorded for 72 of the 77 patients in the I group. Twelve patients (16.5%) had a strongly positive response; 12 (16.5%) had a weakly positive response; 18 (25%) had a positive result; and 30 (42%) patients failed to show an immune response. Thus, 42 (58%) patients in total exhibited a positive effect.
QOL was recorded for 72 of the 77 patients in the I group. Of these, 9 (13%) patients exhibited improvements in all QOL events; 13 (18%) exhibited improvements in 3 events; another 13 (18%) exhibited improvements in 2 events; 12 (16%) exhibited improvement in 1 event; and 25 (35%) exhibited no improvements of the QOL factors.
Adverse effects were assessed in 72 of the 77 patients in the I group. Of these, 37 (51%) did not have fever or cold symptoms and 5 (7%) developed cold symptoms. A total of 30 patients developed fever: Low fever occurred in 10 (14%), moderate fever in 11 (15%) and high fever in 9 patients (13%). A total of 12 patients (17%) developed anorexia; 18 (25%) developed insomnia; 22 (31%) developed arthralgia; and 8 (11%) developed skin rash (located at the intradermal injection site in 7 cases and on the upper as well as the lower body in 1 case).
In conclusion, these findings demonstrated that autoimmune cell therapy is a viable treatment option for HPC patients. The QOL and overall survival of these patients were improved, without severe adverse effects. The mortality rate was lower in the I group compared with that in the NI group and the overall survival was prolonged, proving that immunotherapy is an effective method for cancer treatment.
Article Reference link: click here
Scientific article publishing date: 10/19/2015
Immucura identifier BSC21_367EN